Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease. There are currently no pharmacological treatments for advanced liver fibrosis. The abnormal proliferation and differentiation of mesenchymal stellate cells into myofibroblasts is the driving force of liver scaring, collagen accumulation, and extracellular matrix remodeling often associated with the increase of portal hypertension. In the last few years the beneficial effects of relaxin peptide treatment have been demonstrated in clinically relevant animal models of liver disease. It has been shown that relaxin suppresses the activation of quiescent hepatic stellate cells and their differentiation into collagen producing myofibroblasts. Additionally, treatment with relaxin caused a significant reduction of portal pressure in rats with induced liver fibrosis. Despite such favorable indications the use of relaxin is complicated due to its short half-life in vivo thus requiring continuous peptide delivery; a potential immune response to the injected peptide; and the high cost of the recombinant peptide. Relaxin signals through its cognate G protein-coupled receptor RXFP1 which is expressed in hepatic stellate cells. We have recently identified a first series of small molecule agonists of RXFP1. These molecules are highly specific potent activators of RXFP1; they have a long in vitro and in vivo half-life, a preferred ADME profile, and low cytotoxicity. The preliminary data suggest that the small molecule suppressed the expression of pro-fibrotic genes in primary human stellate cells while increasing expression of genes participating in extracellular matrix degradation. The current application is designed to validate the therapeutic anti-fibrotic effects of RXFP1 agonists using unique in vitro and in vivo models of liver fibrosis. First, we will test the efficacy of lead compounds to suppress fibrosis in activated human stellate cells and test their effects on various liver cells. The selected compounds will be tested in liver mini-organoids, the complex fully functional mini-organs produced for high-throughput analysis of therapeutic drug targets at the Wake Forest Institute for Regenerative Medicine. The most efficacious agonist with low cytotoxicity and no effects on normal hepatic cell function will be selected for animal testing. As RXFP1 agonists do not activate rodent receptors we have produced an RXFP1 humanized strain of mice with the endogenous mouse relaxin receptor replaced by a fully functional human receptor. Using this model we will test the anti-fibrotic properties of relaxin agonists in a toxic chemical liver CCl4 insult model and in a cholestatic liver injury model. These two models reflect different ontogeny and localization of liver fibrosis in humans. The results of our experiments will provide a foundation for future translational studies of relaxin receptor agonists for treating fibrosis in liver and other organs.